The Secretory Component (SC) is a component of secretory immunoglobulin (SIgA and SIgM), comprising the extracellular part of the polymeric immunoglobulin receptor (pIgR). Polymeric IgA and IgM binds, mediated by the J-chain, to the polymeric immunoglobulin receptor on the basolateral surface of epithelial cells and is taken up into the cell via transcytosis. The receptor-immunoglobulin complex passes through the cellular compartments before being secreted on the luminal surface of the epithelial cells, still attached to the receptor. Proteolysis of the receptor occurs and the dimeric IgA molecule or the IgM, along with the Secretory Component, are free to diffuse throughout the lumen.
The Secretory Component has been described to occur in various body secretions such as saliva, tears, mucus and milk. It can be found either as part of secretory immunoglobulins (Sig, i.e. SIgA and SIgM) as well as free Secretory Component (fSC).
Human Secretory Component (hSC) is derived from the polymeric immunoglobulin receptor by cleavage of the extracellular part of the receptor molecule in the process of transcytosis. It has an apparent molecular weight of about 80 kDa and consists of the first about 585 amino acids of the pIgR (polymeric Ig-Receptor) arranged in five V-type immunoglobulin domains. It has 7 potential N-glycosylation sites. This strong glycosylation contributes to the large apparent molecular weight. The composition of these glycans includes bi- and triantennarry structures, Lewis type structures as well as galactose and sialic acids. These glycans constitute binding epitopes for bacterial, viral, fungal and protozoan structures such as adhesins and toxins as well as mucins and receptors on host tissues.
A proporsed function of the Secretory Component is the protection of polymeric immunglobulin from proteolytic degradation and binding to pathogen related structures and toxins such as Helicobacter pylori, enteropathogenic E. coli, Clostridium difficile toxin A and Streptococcus pneumoniae cholin binding protein A. Glycans on SC have proven to participate in innate protection against mucosal pathogens (Perrier et al. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 281(20), pp. 14280-14287, 2006). The authors reported that recombinant human SC produced from transfected Chinese Hamster Ovary cells (CHO) behaved identically to the SC purified from human milk. The interaction with pathogen antigens was mediated by glycans present on hSC and involved galactose and sialic acid residues. hSC was identified as a microbial scavenger contributing to the antipathogenic arsenal that protects the body epithelial surface.
SC purified from human milk was found to competitively inhibit Clostridium difficile toxin A binding to receptors (Dallas et al. J. Med. Microbiol. 47: 879-888 (1998)). Removing carbohydrates from SIgA and SC by enzymatic digestion showed that Clostridium difficile toxin A binds much less to deglycosylated SC than to glycosylated SC.
Human SC presents a wide range of glycan structures, including all of the different Lewis and sialyl-Lewis epitopes that can potentially bind lectins and bacterial adhesins. One can find galactose linked both beta1-4 and beta1-3 to GlcNAc; fucose linked alpha1-3 and alpha1-4 to GlcNAc and alpha1-2 to galactose, as well as both alpha2-3- and alpha2-6-linked sialic acids. More than 50% of the glycans of SC from human milk show various types of non-core fucosylation, the most abundant fucose-containing antigen is Lewis x. About 30% of Lewis-type fucosylated antigens in human SC are sialylated. Altogether, SC from human milk displays more than 50 different glycoforms.
Proposed functions of glycans on SC are e.g.                mediation of anchoring of secretory immunoglobulin in the mucus,        mediation of binding of secretory immunoglobulin/antigen complexes to certain receptors (e.g. DC-SIGN),        the action as competitive inhibitor (“decoy”) of pathogen structure binding to host cells, e.g. by acting as decoy for lectin-like receptors expressed by pathogenic toxins, viruses and bacteria.        the protection of secretory immunoglobulin and of SC against proteases        
The multitude of glycan structures found in natural SC may reflect the multitude of functions. However for certain therapeutic and prophylactic uses of secretory immunoglobulins it may be advantageous to reduce the complexity of the glycan population in a preparation of secretory immunoglobulin. A bias towards certain glycan structures and modifications may increase efficacy and/or reduce potential side effects of such a preparation of secretory immunoglobulin.
Human Secretory Component has been recombinantly expressed in a variety of genetically engineered organisms and cells such as bacteria (E. coli), insect cells (Sf9 cells), mammalian cells (Chinese hamster ovary cells, African green monkey kidney CV-1 cells, human osteosarcoma cells TK-143B, human HeLa cells, Baby hamster kidney cells, human adenocarcinoma cells HT29, mouse fibroblasts, Madin-Darby canine kidney cells) and plants (e.g. US20080260822, EP799310, Michetti et al. 1991 Adv Exp Med Biol, vol 310, pp 183-5; Suguro et al. 2011, Protein Expr Purif. Vol. 78, pp 143-8; Prinsloo et al. 2006 Protein Expr Purif. vol 47, pp 179-85; Ogura, 2005, J Oral Sci. vol 47, pp 15-20; Matsumoto et al. 2003 Scand J Immunol. Vol 58, pp 471-6; Johansen et al. 1999 Eur J Immunol., Vol 29, pp 1701-8; Chintalacharuvu and Morrison 1999, Immunotechnology. Vol 4, pp 165-74; de Hoop et al. 1995. J Cell Biol. Vol 130, pp 1447-59; Larrick et al. 2001 Biomol Eng. Vol 18, pp 87-94; Berdoz et al. 1999, Proc Natl Acad Sci USA. vol 96, pp 3029-34; Rindisbacher et al. 1995, J Biol Chem vol. 270, pp 14220-8).
The glycan pattern found on recombinant SC is dependent on the host species, the host organism, the tissue of origin and the physiological state of the genetically engineered cell.
While E. coli is unable to produce N-glycosylated proteins at all, most of the other hosts used so far for expressing recombinant SC are unable to produce Lewis x fucosylation (Sf9 cells, plant cells, HeLa, CHO cells, CV-1 cells, 143B cells, BHK cells, mouse fibroblasts, MDCK cells). Under the conditions described, HT-29 do not efficiently produce Lewis antigens on N-Glycans. HT-29 cells cultured in glucose have properties of undifferentiated multipotent transit cells, are very unstable, the conversion of high-mannose to complex glycoproteins is, however, severely reduced in HT-29 cells grown in differentiation non-permissive conditions (HT-29 Glc+) whatever the phase of growth studied.
Carbohydrate epitopes in breast milk are known to differ between species, with human milk expressing the most complex one. Gustafsson et al. (Glycoconjugate 22: 109-118 (2005)) investigated the expression of protein-bound carbohydrate epitopes in individual milk samples from man, cow, goat, sheep, pig, horse, dromedary and rabbit.
The glycan pattern found on SC is dependent on the host species, host organism, tissue of origin, and physiological status of the organism (e.g. health status, lactation phase).
Xu et al. (World J. Gastroenterol. 10(14) 2063-2066 (2004)) analysed the effects of fucosylated milk obtained from a transgenic goat. Human alpha1-2/4 fucosyltransferase gene was transiently expressed in goat mammary gland to produce “humanized” goat milk. The goat milk samples were found to inhibit bacterial binding to Lewis b antigen.
WO95/24495A1 describes the transgenic production of oligosaccharides and glycoconjugates in milk of transgenic mammals expressing human glycosyltransferase.
Grabenhorst et al. (Glycoconjugate Journal 1999, 16(2): 81-97) describe the genetic engineering of recombinant glycoproteins in frequently used host cells, e.g. transfection of CHO cells with gylcosyltransferases.
Porter P (Immunology 1973, 24(1) 163-176) describes the purification of porcine Secretory Component and secretory IgA from sow milk.
Gustafsson et al. (Glycoconjugate Journal 2005, 22(3) 109-118) describe the Lewis-type N-glycosylation of human and animal milk proteins.
The pIgR-pIg complex is transcytosed across the cell, and at the luminal surface the pIgR is cleaved by protease within a 42-amino acid region adjacent to the cell membrane thus releasing SIg into the lumen.
The cleaved extracellular portion of pIgR remains bound to pIg and is herein termed Secretory Component (SC). pIgR can also be transported into the mucosa even if pIg is not bound to it, thus most exocrine fluids contain SC both bound within SIg and also free SC.
The precise cleavage site is still ambiguous, as human SC was found to have a ragged C-terminus, varying from Ala-550 to Lys-559, with Ser-552 as the dominant C-terminal residue.
It is possible that additional proteolysis can occur after cleavage of the pIgR. The fact that free SC from different mucosal fluids appear to have slightly different molecular weights might suggest that proteolysis does occur in vivo after release of free SC from pIgR.
Free SC from colostrum has a molecular mass of approximately 76.5 kDa compared with approximately 80 kDa for bound SC to dIgA. The difference has been shown to stem from a difference in length of the polypeptide chain. There is, however, not a clear consensus about the C-terminal end of milk derived SC bound to polymeric immunoglobulin.
The generally larger size of SC in SIgA1 and SIgA2 compared to free SC may result from the presence of dimeric IgA in the former, which may shield the C-terminal linker of SC when the pIgR is cleaved after transcytosis. This shielding by dimeric IgA would be absent when free SC is cleaved in similar circumstances.
Human colostrum and milk are rich in both proteases and protease inhibitors. The ratio of inhibitor to protease defines whether active protease is present. The ratio changes markedly with the time after birth and appears to differ in different individuals. Since the free SC C-terminal linker peptide is highly susceptible to proteases, it might well be the case that there is no “correct” C-terminus for colostral and milk free SC. Moreover, the C-terminus for SC from other tissues than mammary gland (such as gut, bronchial, nasal tissues) may show different C-termini either because of different enzymatic cleavage from pIgR or because of the presence of different proteases trimming the free SC or pIg-bound SC.
One of the most important molecules for protection against infection of humans at mucosal sites (eyes, nose, mouth, lung, ears, traches, esophagus, gastric tract, intestine, urogenital tract and colon) is secretory IgA which may act both via its four antigen binding sites as well as via the glycan mediated binding of the Secretory Component.
Many studies demonstrate strong correlations between titers of specific SIgA antibodies in secretions and resistance to infection. Some studies demonstrate protection against systemic challenge with capsule forming bacterial pathogens.
Saliva and colostrum from normal subjects contain polyreactive SIgA antibodies which recognize a variety of autoantigens and several bacterial antigens. It has been suggested that these are products of B-1 cells, constituting part of the “natural antibody” repertoire encoded in the germline, and lacking memory capability and affinity maturation. These antibodies may provide protection of the mucosal surfaces prior to the generation of specific antibodies from conventional B-2-cells after exposure to nominal antigens. Although they have low intrinsic affinity for antigens, the presence of four antigen-binding sites in SIgA increases its functional activity. There is indeed evidence which suggests that bacterial adhesins have evolved because they are able to avoid recognition by these naturally occurring polyreactive antibodies.
In humans there are two unique IgA-subclasses (IgA1 and IgA2). Two or three allotypes of human IgA2 have been described (different combinations of constant region domains of the alpha-heavy chains). The predominant molecular form of circulating (plasma) IgA is monomeric, in contrast to the dimeric (polymeric) pIgA produced in epithelium and transported into the secretions as SIgA.
Human IgA1 and IgA2 (including allotypes) appear to have few, if any, distinct biologically properties but a notable exception is seen in the differences between IgA subclasses in their susceptibility to bacterial proteases. IgA1 and IgA2 also differ in the distribution of antibody specificities.
Immunization of adults with protein antigens elicits mainly IgA1 and immunization with polysaccharides provokes mainly an IgA2 antibody response. Of the immunoglobulin isotypes that reach mucosal surfaces, SIgA is one of the most stable and this stability has been largely ascribed to SC which masks potential cleavage sites within the Fc-portion.
The specificity of SIgA antibodies for surface structures of microbial surfaces to inhibit adherence to pharyngeal, intestinal, genitourinary tract and gingival epithelia was demonstrated. In addition to a specific, antibody-mediated inhibition of adherence, human IgA, and SIgA in particular, bind to many bacterial species and antigens by means of their carbohydrate chains. A notable example of this is seen in the case of IgA2, which can agglutinate E. coli by a mechanism involving the type I (mannose dependent) pili and type I pilus-dependent adherence of E. coli to epithelial cells.
IgM in external secretions is also associated with a Secretory Component (secretory IgM, SIgM) resulting from its transport into secretions by the pIgR. The concentration of SIgM is lower than that of SIgA either because of the lower proportion of IgM-producing cells in mucosal tissues or because IgM may be less well transported than pIgA due to a molecular weight restriction in pIgR-dependent transport.
Natural antibodies, by definition, are produced in the apparent absence of antigenic stimulation. They are produced by a specific subset of B-cells and do not extensively affinity mature. Natural antibodies of the classes IgA, IgM and IgG have been described. These antibodies are encoded usually by germline genes with few, if any, mutations and have in many cases broad reactivity against PAMPs (pathogen-associated molecular pattern), tumor antigens and a number of autoantigens. Because of their low affinity and germ-line configuration, such polyreactive antibodies do not appear to be true autoantibodies and certainly do not fit into the same category as antigen-specific, somatically-mutated, high affinity pathological autoantibodies.
Natural antibodies are considered as part of the innate immune system. They have been proposed for certain therapeutic uses, e.g. cancer therapy or in infectious diseases.
Many of the polyreactive antibodies have a germ-line or near germ-line sequence and are primarily IgM, but some are also IgG and IgA.
Contrary to the classic “lock and key” rigid structure hypothesis of antigen-antibody interaction, the antigen binding pocket of polyreactive antibodies, perhaps because of their germ-line configuration, are believed to be more flexible and therefore can accommodate different antigenic configurations.
Although some reports have suggested that SIgA is polyreactive in nature, other findings point to a restricted specificity that may be cross-reactive.
WO2009139624A1 discloses a process for producing compositions that are rich in secretory IgA by fractionating non-human milk. Such compositions may be used in particular for treating and/or preventing infections and/or inflammation of the mucosal surfaces, e.g. the gastro-intestinal tract, urogenital tract, respiratory tract, nasal cavity or oral cavity, treating and/or preventing obesity and related diseases, or treating and/or preventing food allergies in subjects in need of such treatment.
It is well known that human milk contains high amounts of Lewis-glycosylated glycoconjugates such as glycoproteins (such as SIgA and SC) and it is well accepted that one of the values of human milk is its high protective potency against infection by antibodies, glycans, oligosaccharides and other active substances such as lysozyme and lactoferrin. While the human milk analysis revealed a 75% fucosylation distribution, only 31% fucosylation distribution was observed in the bovine milk analysis. Only core fucosylation has been detected in the bovine milk analysis.
Though human secretory immunoglobulins with certain glycosylation have proven an advantageous effect with respect to binding to pathogen structures, mucins and receptors, they could not yet been produced on a large scale in a desirable quality. For ethical reasons human milk is typically not considered as a suitable source material.